Identification of Trichoderma Species Using Partial Sequencing of nrRNA and tef1α Genes with Report of Trichoderma capillare in Iran Mycoflore

Document Type : Research Article

Authors

• mehdi Mehrabi-Koushki ¹
• Maryam Bavarsad ²
• Reza Farrokhi-Nejad ³

¹ Shahid Chamran University of Ahvaz

² Shahid Chamran university of Ahvaz

³ Shahid Chamran university of ahvaz

10.22067/jpp.v31i3.48738

Abstract

Introduction: Trichoderma is monophyletic (16), with teleomorphs in the genus Hypocrea. Some cryptic Trichoderma species are hidden within morphological species complexes and can only be elucidated by in-depth molecular studies. The genealogical concordance phylogenetic species recognition (GCPSR) using several non-linked genes are needed to give accurate identification of Trichoderma spp. (6). Although the ITS region has been successfully used for species delimitation of Trichoderma and Hypocrea (5), but, it is not sufficient for accurate identification of some species. Translation elongation factor 1α gene (tef1α) is a reliable barcode for Fusarium (9), Trichoderma and Hypocrea (5). Here, ITS and tef1α genes were selected as candidate DNA barcodes to identify Trichoderma isolates.
Material and methods: 40 Trichoderma isolates used in this study were from a fungal collection archived in the plant pathology laboratory in the Department of Plant Protection at the Shahid Chamran University of Ahvaz. Spore suspension (105/ml) prepared from single spore cultures of each Trichoderma isolates was added into flasks containing PDB medium. The flasks were shaken at 180 rpm for 10-15 days at 28ºC and the biomass was harvested by passing through sterilized filter papers. The mycelia were freeze-dried (Freeze-Dryer, Alpha 1-2LD Plus, Christ) and powdered in the mortar containing liquid nitrogen by pestle. The genomic DNA was isolated according to modified method established by Raeder and Broda (21). The universal primers (ITS1–F; 5'-TCCGTAGGTGAACCTGCGG-3' and ITS4-R; 5'-TCCTCCGCTTATTGATATGC-3') were employed for amplifying around 700bp from 18s, ITS1, 5.8s, ITS2 and 28s rDNA regions (27). The specific primers (tef1α71-f; 5'-CAAAATGGGTAAGGAGGASAAGAC-3' and tef1997-R; 5'-CAGTACCGGCRGCRATRATSAG-3') were employed for amplifying around 950bp from tef1α gene (24). PCR products were purified through ethanol-precipitation method and then sequenced using forward and reverse primers by Macrogen Company. The Sequences were edited and assembled using BioEdit v. 7.0.9.0 (10) and DNA Baser Sequence Assembeler v4 programs (2013, Heracle BioSoft, www.DnaBaser.com), respectively. These sequences were submit-queried against the NCBI non-redundant database and related to known DNA sequences by BLASTn algorithm to assign putative identity. They also were subjected to the TrichO Key (5) and TrichoBLAST (15) for more characterization. The phylogenetic tree was constructed through maximum likelihood analysis based on tef1α sequence under K2+G model. The tree was rooted to close species of N. macroconidialis.
Result and Discussion: Approximately 550 and 850 bases of the ITS and tef1α regions were sequenced from the isolates studied and then deposited in the GenBank (Table 2). The annotation of indexed sequences showed which multiple insertion-type frame shifts have interestingly occurred into the reading frame of tef1α gene belonging to isolate of T. capillare Isf-7 (Fig. 3). To identify isolates of Trichoderma, ITS and tef1α sequences were subjected to the TrichO Key (5), TrichoBLAST (15) and BLASTn Search. The analysis of ITS and tef1α sequences (Table 2, Fig. 2), in combination with morphology (Table 1), showed which the isolates place in seven species as follow: T. harzianum Rifai, T. virens (J.H. Mill., Giddens & A.A. Foster) Arx, T. pleuroticola Yu & Park, T. asperellum Samuels, Lieckf & Nirenberg, T. koningiopsis Oudem., T. brevicompactum Kraus, Kubicek & Gams and T. capillare Samuels & Kubicek. In BLASTn search, ITS and tef1α regions separately provided unambiguous identification for isolates of T. virens, T. koningiopsis and T. brevicompactum while ITS region provided ambiguous identification for Isolates of Trichoderma harzianum, T. capillare, T. pleuroticola and T. asperellum. Here, tef1α region could provide more accurate identification as good DNA barcoding (Table 2). The isolates showed the sequence identity ranging from 96 to 100% for tef1α locus and 88 to 99% for ITS locus. Different identities related to ITS and tef1α genes indicated that the single gene identification is not accurate, particularly for Trichoderma species, if the identification is based on ITS regions (4). In phylogenetic tree (Fig. 2), the isolates surveyed generated strongly supported clades for each species, distinct from other species. Among the species identified, T. capillare is the first report for Iran mycoflora. This species was firstly described by Samuels et al. (22) and phylogenetically delimited from other species of Longibrachiatum section.
Conclusions: Here, of seven species of Trichoderma identified, the species of T. capillare is newly reported in Iran. Our studies demonstrate ultimately that, despite ITS region, tef1α gene is quite reliable in identification and phylogeny of Trichoderma species.


Material and methods: 40 Trichoderma isolates used in this study were from a fungal collection archived in the plant pathology laboratory in the Department of Plant Protection at the Shahid Chamran University of Ahvaz. Spore suspension (105/ml) prepared from single spore cultures of each Trichoderma isolates was added into flasks containing PDB medium. The mycelia were harvested from the growth medium by washing biomass with sterilized distilled water on filter papers. Mycelial biomasses were freeze-dried and then powdered into mortar containing liquid nitrogen by pestle. The genomic DNA was isolated according to modified method established by Reader and Broda (1985). The universal primers (ITS1–F; 5'-TCCGTAGGTGAACCTGCGG-3' and ITS4-R; 5'-TCCTCCGCTTATTGATATGC-3') were employed for amplifying around 700bp from 18s, ITS1, 5.8s, ITS2 and 28s rDNA regions. The specific primers (tef1α71-f; 5'-CAAAATGGGTAAGGAGGASAAGAC-3' and tef1997-R; 5'-CAGTACCGGCRGCRATRATSAG-3') were employed for amplifying around 950bp from exon1 to exon6 regions of tef1α gene containing introns 1 to 5 (Shoukouhi and Bisset, 2008). PCR products were purified through ethanol-precipitation method and then sequenced using forward and reverse primers by Macrogen Company. The Sequences were edited and assembled using BioEdit v. 7.0.9.0 (Hall 1999) and DNA Baser Sequence Assembeler v4 programs (2013, Heracle BioSoft, www.DnaBaser.com), respectively. These sequences were submit-queried against the NCBI non-redundant database and related to known DNA sequences by BLASTn algorithm to assign putative identity. They also were subjected to the TrichO Key (Druzhinina et al. 2005) and TrichoBLAST (Kopchinskiy et al. 2005) for more detection.

Result and Discussion: Approximately 550 and 850 bases of the ITS and tef1α regions were sequenced from the isolates studied and then deposited in the GenBank. There was no ITS sequence of T. capillare in databases and we here indexed it and more sequence from its tef1α gene in GenBank. The annotation of indexed sequences showed which multiple insertion-type frame shifts have interestingly occurred into reading frame of tef1α gene belong to T. capillare Isf-7 isolate (Fig. 1). To identify isolates of Trichoderma, ITS and tef1α sequences were subjected to the TrichO Key (Druzhinina et al. 2005), TrichoBLAST ( Kopchinskiy et al. 2005) and BLASTn Search. The analysis and comparison of ITS and tef1α data with reference sequences in ISTHT and GenBank showed which the isolates place in seven species as follow: T. harzianum Rifai, T. virens (J.H. Mill., Giddens & A.A. Foster) Arx, T. pleuroticola Yu & Park, T. asperellum Samuels, Lieckf & Nirenberg, T. koningiopsis Oudem., T. brevicompactum Kraus, Kubicek & Gams and T. capillare Samuels & Kubicek. In BLASTn search, ITS and tef1α regions separately provided unambiguous identification for isolates of T. virens, T. koningiopsis and T. brevicompactum while ITS region provided ambiguous identification for Isolates of Trichoderma harzianum, T. capillare Samuels & Kubicek, T. pleuroticola and T. asperellum. Here, tef1α region could provide more accurate identification as good DNA barcoding (Table 2). The isolates showed the sequence identity ranging from 96 to 100% for tef1α locus and 88 to 99% for ITS locus. Different identities related to ITS and tef1α genes indicated that single gene identification is not accurate, particularly for Trichoderma species, if the identification is based on ITS regions (Druzhinina and Kubicek, 2005). Among the species identified, T. capillare is the first report for Iran mycoflora. This species was firstly described by Samuels et al. (2012) and phylogenetically associated with other species of Longibrachiatum Clade.

Conclusions: Here, of seven species of Trichoderma identified, the species of T. capillare is newly reported in Iran. Our studies demonstrate ultimately that, despite ITS region, tef1α gene is quite reliable in identification and phylogeny of Trichoderma species.

Keywords

• DNA-barcoding
• morphology
• phylogenetic clustering

20.1001.1.20084749.1396.31.3.1.2

---

References

1- Ainsworth G.C. 1971. Ainsworth and Bisby’s dictionary of the fungi. 6th ed. Common wealth Mycological Institute, Kew, Surrey, England.

2- Crouse J., and Amorese D. 1987. Ethanol precipitation: ammonium acetate as an alternative to sodium acetate. Focus, 9: 3–5.

3- Dodd S., Crowhurst R.N., Rodrigo A.G., Samuels G.J., Hill R.A., and Stewart A. 2003. Examination of Trichoderma phylogenies derived from ribosomal DNA sequence data. Mycological Research, 4: 32-39.

4- Druzhinina I., and Kubicek C.P. 2005. Species concepts and biodiversity in Trichoderma and Hypocrea: from aggregate species to species clusters. Journal of Zhejiang University Science Botany, 6: 100-112.

5- Druzhinina I.S., Kopchinskiy A.G., Komon M., Bissett J., Szakacs G., and Kubicek C.P. 2005. An oligonucleotide barcode for species identification in Trichoderma and Hypocrea. Fungal Genetics and Biology, 42: 813-828.

6- Druzhinina I.S., Kopchinskiy A.G., and Kubicek C.P. 2006. The first one hundred of Trichoderma species is characterized by molecular data. Mycoscience, 47: 55-64.

7- Druzhinina I.S., Komo -Zelazowska M., Ismaiel A., Jaklitsch W., Mullaw T., Samuels G.J., and Kubicek C.P. 2012. Molecular phylogeny and species delimitation in the section Longibrachiatum of Trichoderma. Fungal Genetics and Biology, 49: 358-368.

8- Gams W., and Bissett J. 1998. Morphology and identification of Trichoderma. pp. 3-31. In C.P. Kubicek and G.E. Harman (eds.) Trichoderma and Gliocladium, Taylor & Francis, London.

9- Geiser D.M., Jimenz Gasco M.M., Kang S., Mkalowska I., Veeraraghavan N., Ward T.J., Zhang N., Kuldau G.A., and O’Donnell K. 2004. Fusarium-IDv.1.0: A DNA sequence database for identifying Fusarium. European Journal of Plant Pathology, 110: 473-479.

10- Hall T.A. 1999. BioEdit: a user-friendly biological sequence alignment editor and analysis program for windows 95/98/NT. Nucleic Acids Symposium Series, 41: 95-98.

11- Harman G.E., Howell C.R., Viterbo A., Chet I., and Lorito M. 2004. Trichoderma species-opportunistic, avirulent plant symbionts. Nature Review Microbiology, 2:43–56.

12- Hebert P.D.N., Cywinska A., Ball S.L., and deWaard J.R. 2003a. Biological identifications through DNA barcodes. Proceedings of the Royal Society of London: Series B, 270: 313–321.

13- Hebert P.D.N., Ratnasingham S., and deWaard J.R. 2003b. Barcoding animal life: cytochrome c oxidase subunit 1 divergences among closely related species. Proceedings of the Royal Society of London: Series B, 270: 96–99.

14- Karimi S. 2005. Help to identification of Trichoderma species and endomycorrhizae as useful fungi of walnut rhizosphere in Hamadan province. MSc thesis, agriculture faculty, Buali Sina University of Hamadan, Iran. (In Persian with English abstract)

15- Kopchinskiy A.G., Komon M., Kubicek C.P., and Druzhinina I.S. 2005. TrichoBLAST: A multilocus database for Trichoderma and Hypocrea identifications. Mycological Research, 109:657–660.

16- Kullnig-Gradinger C., Szakacs G., and Kubicek C.P. 2002. Phylogeny and evolution of the genus Trichoderma: a multigene approach. Mycological Research, 106:757–767.

17- Mehrabi-Koushki M., Rouhani H., and Farsi M. 2011. Genetic manipulation of fungal strains for Improvement of heterologous genes expression (a minireview). African Journal of Biotechnology, 10: 7939-7948.

18- Mehrabi-Koushki M., Rouhani H., and Mahdikhani-Moghaddam E. 2012. Differential display of abundantly expressed genes of T. harzianum during colonization of tomato-germinating seeds and roots. Current Microbiology, 65:524–533.

19- Naeimi S., Khodaparast S.A., Javan-Nikkhah M., Vagvolgyi. C., and Kredics L. 2011. Species patterns and phylogenetic relationships of Trichoderma strains in rice fields of southern Caspian sea, Iran. Cereal Research Communications, 39: 560–568.

20- Nazmi-Roodsari F., Zafari D., Khodaparast A., and Rouhani H. 2007. Introducing some new species of Trichoderma for Iran. Rostaniha, 8:67-88.

21- Raeder U., and Broda P. 1985. Rapid preparation of DNA from filamentous fungi. Letters in Applied Microbiology, 1: 17–20.

22- Samuels G.J., Ismaiel A., Mullaw T.B., Szakacs G., Druzhinina I.S., Kubicek C.P., and Jaklitsch W.M. 2012. The Longibrachiatum Clade of Trichoderma: a revision with new species. Fungal Diversity, 55:77-108.

23- Samuels G.J., Chaverri P., Farr D.F., and McCray E.B. 2016. Trichoderma Online, Systematic Mycology and Microbiology Laboratory, ARS, USDA, available from: http://nt.ars-grin.gov/taxadescriptions/keys/TrichodermaIndex.cfm.

24- Shoukouhi E., and Bissett J. 2008. Preferred primers for sequencing the 50 end of the translation elongation factor 1-alpha gene (EF1-a1) and subunit 2 of the RNA polymerase B gene (RPB2). ISTH available from: http://www.isth.info/methods.

25- Tamura K., Stecher G., Peterson D., Filipski A., and Kumar S. 2013. MEGA6: molecular evolutionary genetics analysis version 6.0. Molecular Biology and Evolution, 30: 2725-2729.

26- Vahabi K. 2004. Genetic diversity of Trichoderma species related to Agaricus bisporus mushroom using morphological and molecular methods. MSc. Thesis, agriculture faculty, Isfahan University of the technology, Iran. (In Persian with English abstract)

27- White T.J., Bruns T., Lee S., and Taylor J. 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. P315–322. In: MA Innis et al. (ed.) PCR Protocols: a guide to methods and applications. Academic Press, New York, USA.

28- Zafari D., Zare R., Ershad D., and Alizadeh A. 2003. A contribution to the identification of Trichderma species in Iran. Iranian Journal of Plant Pathology, 38: 9-15. (In Persian with English abstract)

29- Zafari D., Zare R., Ershad D., and Alizadeh A. 2004. Introduction of three new species of Trichoderma for mycoflora of Iran. Rostaniha, 5: 63-65. (In Persian with English abstract)